Suppressed immune microenvironment and repertoire in brain metastases from patients with resected non-small-cell lung cancer.

BACKGROUND: The tumor immune microenvironment (TIME) of lung cancer brain metastasis is largely unexplored. We carried out immune profiling and sequencing analysis of paired resected primary tumors and brain metastases of non-small-cell lung carcinoma (NSCLC). PATIENTS AND METHODS: TIME profiling of archival formalin-fixed and paraffin-embedded specimens of paired primary tumors and brain metastases from 39 patients with surgically resected NSCLCs was carried out using a 770 immune gene expression panel and by T-cell receptor beta repertoire (TCRß) sequencing. Immunohistochemistry was carried out for validation. Targeted sequencing was carried out to catalog hot spot mutations in cancer genes. RESULTS: Somatic hot spot mutations were mostly shared between both tumor sites (28/39 patients; 71%). We identified 161 differentially expressed genes, indicating inhibition of dendritic cell maturation, Th1, and leukocyte extravasation signaling pathways, in brain metastases compared with primary tumors (P < 0.01). The proinflammatory cell adhesion molecule vascular cell adhesion protein 1 was significantly suppressed in brain metastases compared with primary tumors. Brain metastases exhibited lower T cell and elevated macrophage infiltration compared with primary tumors (P < 0.001). T-cell clones were expanded in 64% of brain metastases compared with their corresponding primary tumors. Furthermore, while TCR repertoires were largely shared between paired brain metastases and primary tumors, T-cell densities were sparse in the metastases. CONCLUSION: We present findings that suggest that the TIME in brain metastases from NSCLC is immunosuppressed and comprises immune phenotypes (e.g. immunosuppressive tumor-associated macrophages) that may help guide immunotherapeutic strategies for NSCLC brain metastases.

Mutation profiles in early-stage lung squamous cell carcinoma with clinical follow-up and correlation with markers of immune function.

Background: Lung squamous cell carcinoma (LUSC) accounts for 20­30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We performed whole-exome sequencing (WES) and comprehensive immune profiling of a unique set of clinically annotated early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. Methods: Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers were done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using analysis of variance and Fisher's exact test. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. Results: This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for somatic mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild-type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. Conclusion(s): Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC.

Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma with fully annotated clinical follow-up.

Background: Lung adenocarcinomas (LUADs) lead to the majority of deaths attributable to lung cancer. We performed whole-exome sequencing (WES) and immune profiling analyses of a unique set of clinically annotated early-stage LUADs to better understand the pathogenesis of this disease and identify clinically relevant molecular markers. Methods: We performed WES of 108 paired stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Ten immune markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry (IHC) in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fisher's exact test. Cox proportional hazards regression models were used for multivariate analysis of clinical outcome. Results: LUADs in this cohort exhibited an average of 243 coding mutations. We identified 28 genes with significant enrichment for mutation. SETD2-mutated LUADs exhibited relatively poor recurrence- free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS among KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis revealed that LUADs in smokers and with relatively high mutation burdens exhibited increased levels of immune markers. Analysis of immunophenotypes revealed that LUADs with STK11 mutations exhibited relatively low levels of infiltrating CD4+/CD8+ T-cells indicative of a muted immune response. Tumoral PD-L1 was significantly elevated in TP53 mutant LUADs whereas PIK3CA mutant LUADs exhibited markedly down-regulated PD-L1 expression. LUADs with TP53 or KEAP1 mutations displayed relatively increased CD57 and Granzyme B levels indicative of augmented natural killer (NK) cell infiltration. Conclusion(s): Our study highlights molecular and immune phenotypes that warrant further analysis for their roles in clinical outcomes and personalized immune-based therapy of LUAD.

An in vivo screening system to identify tumorigenic genes.

Screening for oncogenes has mostly been performed by in vitro transformation assays. However, some oncogenes might not exhibit their transforming activities in vitro unless putative essential factors from in vivo microenvironments are adequately supplied. Here, we have developed an in vivo screening system that evaluates the tumorigenicity of target genes. This system uses a retroviral high-efficiency gene transfer technique, a large collection of human cDNA clones corresponding to ~70% of human genes and a luciferase-expressing immortalized mouse mammary epithelial cell line (NMuMG-luc). From 845 genes that were highly expressed in human breast cancer cell lines, we focused on 205 genes encoding membrane proteins and/or kinases as that had the greater possibility of being oncogenes or drug targets. The 205 genes were divided into five subgroups, each containing 34-43 genes, and then introduced them into NMuMG-luc cells. These cells were subcutaneously injected into nude mice and monitored for tumor development by in vivo imaging. Tumors were observed in three subgroups. Using DNA microarray analyses and individual tumorigenic assays, we found that three genes, ADORA2B, PRKACB and LPAR3, were tumorigenic. ADORA2B and LPAR3 encode G-protein-coupled receptors and PRKACB encodes a protein kinase A catalytic subunit. Cells overexpressing ADORA2B, LPAR3 or PRKACB did not show transforming phenotypes in vitro, suggesting that transformation by these genes requires in vivo microenvironments. In addition, several clinical data sets, including one for breast cancer, showed that the expression of these genes correlated with lower overall survival rate.

Prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia: a report from the Japanese Pediatric Leukemia/Lymphoma Study Group.

CCAAT/enhancer-binding protein alpha (CEBPA) mutations are a favorable prognostic factor in adult acute myeloid leukemia (AML) patients; however, few studies have examined their significance in pediatric AML patients. Here we examined the CEBPA mutation status and clinical outcomes of pediatric AML patients treated in the AML-05 study. We found that 47 (14.9%) of the 315 evaluable patients harbored mutations in CEBPA; 26 cases (8.3%) harbored a single mutation (CEBPA-single) and 21 (6.7%) harbored double or triple mutations (CEBPA-double). After excluding core-binding factor-AML cases, patients harboring CEBPA mutations showed better overall survival (OS; P=0.048), but not event-free survival (EFS; P=0.051), than wild-type patients. Multivariate analysis identified CEBPA-single and CEBPA-double as independent favorable prognostic factors for EFS in the total cohort (hazard ratio (HR): 0.47 and 0.33; P=0.02 and 0.01, respectively). CEBPA-double was also an independent favorable prognostic factor for OS (HR: 0.30; P=0.04). CEBPA-double remained an independent favorable factor for EFS (HR: 0.28; P=0.04) in the normal karyotype cohort. These results suggest that CEBPA mutations, particularly CEBPA-double, are an independent favorable prognostic factor in pediatric AML patients, which will have important implications for risk-stratified therapy.

Value of pre- and postoperative meticillin-resistant Staphylococcus aureus screening in patients undergoing gastroenterological surgery.

BACKGROUND: Data supporting active surveillance of meticillin-resistant Staphylococcus aureus (MRSA) for the prevention of postoperative infection remain controversial. AIM: To investigate the efficacy of MRSA screening in patients undergoing gastroenterological surgery. METHOD: Nasal carriage of MRSA was screened using a polymerase chain reaction (PCR) assay on two gastroenterological surgery wards (A and B). Occurrence of postoperative MRSA infection was analysed according to nasal MRSA carriage status (pre-operative carriage and postoperative acquisition). FINDINGS: The incidence of pre-operative MRSA carriage was 9.7% on Ward A and 4.3% on Ward B (P = 0.009). Postoperative nasal MRSA acquisition was confirmed in 16.2% and 6.0% of patients, respectively (P < 0.001). There was no significant difference in the incidence of MRSA surgical site infections (SSIs) between patients with and without pre-operative nasal colonization on either ward. On Ward A, where MRSA nasal acquisition was more common, the MRSA infection rate in patients with postoperative nasal acquisition was 26.8%, which was significantly higher than the rates in patients with pre-operative MRSA colonization and patients without colonization during hospitalization. Postoperative nasal MRSA acquisition was an independent factor associated with MRSA infection on both wards [Ward A: odds ratio (OR) 7.192, 95% confidence interval (CI) 2.981-17.352; Ward B: OR 5.761, 95% CI 1.429-23.220]. CONCLUSION: MRSA SSIs were prevented by a screening-based strategy in pre-operative MRSA carriers. Postoperative nasal acquisition was a significant factor affecting MRSA infection, and the effect of screening varied according to the incidence of postoperative MRSA acquisition on the ward.

Interferon γ and plasminogen activator inhibitor 1 regulate adhesion formation after partial hepatectomy.

BACKGROUND: The pathophysiology of intra-abdominal adhesions has not been studied extensively. The aim of this study was to elucidate the molecular mechanisms underlying adhesion formation in a murine model and in patients undergoing hepatectomy. METHODS: Partial hepatectomy was performed using bipolar forceps in mice. Wild-type mice, antibodies to CD4 and interferon (IFN) γ, IFN-γ, natural killer T (NKT) cells and plasminogen activator inhibitor (PAI) 1 knockout (KO) mice were used. Recombinant hepatocyte growth factor (HGF) was tested for its ability to prevent adhesions. Liver specimens were obtained during surgery from patients undergoing hepatectomy. Adhesion formation was evaluated using a scoring system that ranged from 0 (no adhesions) to 5 (severe adhesions). Levels of IFN-γ and PAI-1 mRNA, and protein concentration of PAI-I were measured, and fluorescence immunostaining was performed. RESULTS: Adhesion formation depended on IFN-γ produced by NKT cells, and NKT KO mice developed few adhesions (mean(s.d.) 1·7(0·3) versus 4·6(0·4) in wild-type mice; P = 0·037). In wild-type mice, the level of PAI-1 mRNA increased after hepatectomy, followed by a decrease in the tissue plasminogen activator (tPA) mRNA level. Adhesion formation was inhibited completely in PAI-1 KO mice (0(0) versus 4·1(0·8) in wild-type mice; P = 0·002). HGF inhibited formation of abdominal adhesions after hepatectomy by reducing IFN-γ and PAI-1 levels, and increasing tPA levels compared with those in mice treated with phosphate-buffered saline (P < 0·001, P = 0·002 and P = 0·035 respectively). In human liver specimens, NKT cells accumulated in the liver after hepatectomy, and PAI-1 expression was increased 5·25-fold (P = 0·030). CONCLUSION: IFN-γ is a key molecule for abdominal adhesion formation after hepatectomy, acting via the reciprocal balance of PAI-1 and tPA. This molecular mechanism may also regulate adhesion formation in patients following hepatectomy. HGF inhibited formation of adhesions by regulating IFN-γ and PAI-1, suggesting that it may be an important target for prevention of adhesions after hepatectomy. SURGICAL RELEVANCE: Postoperative intra-abdominal adhesions can be asymptomatic or cause significant morbidity and mortality. Adhesion formation after hepatectomy has not been studied extensively. In the present study, the molecular mechanisms underlying intra-abdominal adhesions after hepatectomy were investigated in a murine model and in patients. Interferon (IFN) γ produced by natural killer T cells is a key molecule for adhesion formation after hepatectomy in mice, acting via the reciprocal balance between plasminogen activator inhibitor (PAI) 1 and tissue plasminogen activator, the pivotal factors in fibrinolytic activity. This mechanism was also involved in the regulation of adhesions in human tissue samples. Hepatocyte growth factor (HGF) strongly inhibited adhesion formation by regulating IFN-γ and PAI-1. These results indicate that IFN-γ and PAI-1 are possible therapeutic targets, and HGF could prevent postoperative adhesion formation after hepatectomy.

Laser capture microdissection for the investigative pathologist.

An important step in translational research is the validation of molecular findings from in vitro experiments using tissue specimens. However, tissue specimens are complex and contain a multitude of diverse cell populations that interfere with the molecular profiling data of a specific cell type. Laser capture microdissection (LCM) alleviates this issue by providing a valuable tool for the enrichment of a specific cell type within complex tissue samples. However, LCM and molecular analysis from tissue specimens can be complex and challenging due to numerous issues related with the tissue processing and its impact on the integrity of biomolecules in the specimen. The intricate nature of this application highlights the essential role a pathologist plays in translational research by contributing an expertise in histopathology, tissue handling, tissue analysis techniques, and clinical correlation of biological findings. The present review examines key practical aspects in tissue handling, specimen selection, quality control, and sample preparation for LCM and downstream molecular analyses that are a primary objective of the investigative pathologist.

Estimation of mouth level exposure to smoke constituents of cigarettes with different tar levels using filter analysis.

A nicotine part-filter method can be applied to estimate smokers' mouth level exposure (MLE) to smoke constituents. The objectives of this study were (1) to generate calibration curves for 47 smoke constituents, (2) to estimate MLE to selected smoke constituents using Japanese smokers of commercially available cigarettes covering a wide range of International Organization for Standardization tar yields (1-21mg/cigarette), and (3) to investigate relationships between MLE estimates and various machine-smoking yields. Five cigarette brands were machine-smoked under 7 different smoking regimes and smoke constituents and nicotine content in part-filters were measured. Calibration curves were then generated. Spent cigarette filters were collected from a target of 50 smokers for each of the 15 brands and a total of 780 filters were obtained. Nicotine content in part-filters was then measured and MLE to each smoke constituent was estimated. Strong correlations were identified between nicotine content in part-filters and 41 out of the 47 smoke constituent yields. Estimates of MLE to acetaldehyde, acrolein, 1,3-butadiene, benzene, benzo[a]pyrene, carbon monoxide, and tar showed significant negative correlations with corresponding constituent yields per mg nicotine under the Health Canada Intense smoking regime, whereas significant positive correlations were observed for N-nitrosonornicotine and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone.

Changes in the expression of the Alzheimer's disease-associated presenilin gene in drosophila heart leads to cardiac dysfunction.

Mutations in the presenilin genes cause the majority of early-onset familial Alzheimer's disease. Recently, presenilin mutations have been identified in patients with dilated cardiomyopathy (DCM), a common cause of heart failure and the most prevalent diagnosis in cardiac transplantation patients. However, the molecular mechanisms, by which presenilin mutations lead to either AD or DCM, are not yet understood. We have employed transgenic Drosophila models and optical coherence tomography imaging technology to analyze cardiac function in live adult Drosophila. Silencing of Drosophila ortholog of presenilins (dPsn) led to significantly reduced heart rate and remarkably age-dependent increase in end-diastolic vertical dimensions. In contrast, overexpression of dPsn increased heart rate. Either overexpression or silencing of dPsn resulted in irregular heartbeat rhythms accompanied by cardiomyofibril defects and mitochondrial impairment. The calcium channel receptor activities in cardiac cells were quantitatively determined via real-time RT-PCR. Silencing of dPsn elevated dIP3R expression, and reduced dSERCA expression; overexprerssion of dPsn led to reduced dRyR expression. Moreover, overexpression of dPsn in wing disc resulted in loss of wing phenotype and reduced expression of wingless. Our data provide novel evidence that changes in presenilin level leads to cardiac dysfunction, owing to aberrant calcium channel receptor activities and disrupted Wnt signaling transduction, indicating a pathogenic role for presenilin mutations in DCM pathogenesis.

Blood flow dynamics of one cardiac cycle and relationship to mechanotransduction and trabeculation during heart looping.

Analyses of form-function relationships during heart looping are directly related to technological advances. Recent advances in four-dimensional optical coherence tomography (OCT) permit observations of cardiac dynamics at high-speed acquisition rates and high resolution. Real-time observation of the avian stage 13 looping heart reveals that interactions between the endocardial and myocardial compartments are more complex than previously depicted. Here we applied four-dimensional OCT to elucidate the relationships of the endocardium, myocardium, and cardiac jelly compartments in a single cardiac cycle during looping. Six cardiac levels along the longitudinal heart tube were each analyzed at 15 time points from diastole to systole. Using image analyses, the organization of mechanotransducing molecules, fibronectin, tenascin C, α-tubulin, and nonmuscle myosin II was correlated with specific cardiac regions defined by OCT data. Optical coherence microscopy helped to visualize details of cardiac architectural development in the embryonic mouse heart. Throughout the cardiac cycle, the endocardium was consistently oriented between the midline of the ventral floor of the foregut and the outer curvature of the myocardial wall, with multiple endocardial folds allowing high-volume capacities during filling. The cardiac area fractional shortening is much higher than previously published. The in vivo profile captured by OCT revealed an interaction of the looping heart with the extra-embryonic splanchnopleural membrane providing outside-in information. In summary, the combined dynamic and imaging data show the developing structural capacity to accommodate increasing flow and the mechanotransducing networks that organize to effectively facilitate formation of the trabeculated four-chambered heart.

Evaluation of the Pentax-AWS(®) and the Macintosh laryngoscope in difficult intubation: a manikin study.

BACKGROUND: The Pentax-AWS (AWS(®)), a new video laryngoscope, has been shown to be useful in cases of difficult intubation. We hypothesized that the AWS(®) would be more useful in the settings of a narrow upper airway than the Macintosh laryngoscope. We compared each device in simulated scenarios of representative difficulty of tracheal intubation using a manikin. The primary endpoint was the rate of successful intubation. METHODS: With each device, 23 anesthesiologists performed tracheal intubation in a SimMan(®) manikin in the following scenarios: (1) normal airway, (2) tongue edema, (3) cervical spine rigidity, (4) pharyngeal obstruction, (5) jaw trismus, (6) tongue edema with pharyngeal obstruction. The intubation time and success rate were measured. Each participant was asked to rate the difficulty of intubation (1=very easy; 5=very difficult). RESULTS: In the scenarios of tongue edema and tongue edema with pharyngeal obstruction, the AWS(®) yielded a higher success rate (100% vs. 34.8%; P<0.001, 65.2% vs. 21.7%; P=0.006), a shorter intubation time [14.6 (7.0) vs. 33.4 (13.0) s; P<0.001, 24.5 (12.0) vs. 37.6 (11.9); P=0.047; mean (standard deviation)], and a lower difficulty score [2 (1-4) vs. 5 (1-5); P<0.001, 4 (2-5) vs. 5 (3-5); P<0.001; median (range)], compared with the Macintosh laryngoscope. CONCLUSION: The AWS(®) has an advantage over the Macintosh laryngoscope in simulated tongue edema and tongue edema with pharyngeal obstruction. Further studies in a clinical setting are necessary to confirm these findings.

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide.

AIMS: To develop a quick and accurate PCR-based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. METHODS AND RESULTS: The number of BbrY in faeces was detected by using strain-specific quantitative real-time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA-intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10(5) -10(9) cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (± 1·5) log CFU g(-1) [mean (± SD)] of BbrY was detected in faeces by using strain-specific transgalactosylated oligosaccharide-carbenicillin (T-CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (± 1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (± 0·8) log cells per g. CONCLUSIONS: Strain-specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. SIGNIFICANCE AND IMPACT OF THE STUDY: Combination of strain-specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.

Novel therapeutic strategy for uterine arteriovenous fistulas: case report.

BACKGROUND: The major presenting symptom of uterine arteriovenous fistulas is massive, torrential vaginal bleeding, the degree of which often leads to a shock state. CASE: A 35-year-old woman, gravida 3, para 2 presented with massive vaginal hemorrhage at the first menstruation six months after delivery. Uterine arteriovenous fistulas were diagnosed by color Doppler ultrasonography (US), dynamic computer tomography (CT), and conventional angiography. The patient underwent hysterectomy after bloodstream decrease by bilateral uterine artery embolization. CONCLUSION: The extent of uterine arteriovenous fistulas was diagnosed by color Doppler US, CT, and pelvic angiography, and this precise evaluation led to an adequate therapeutic strategy for uterine arteriovenous fistulas.

Trends in cancer mortality in the elderly in Japan, 1970-2007.

OBJECTIVE: The primary objective of this study is to describe cancer mortality rates and trends among Japanese elderly aged 65-84 years for the period 1970-2007. MATERIALS AND METHODS: Age-standardized mortality rates were calculated by the direct method using age-specific mortality rates at 5-year age intervals and weights based on the age distribution of the standard world population. The joinpoint regression model was used to describe changes in trends. RESULTS: For all cancers combined, the mortality rate at age 65-84 years during 2000-2007 was 1145.13 (per 100,000 population) for men and 461.93 (per 100,000) for women. Mortality rates have declined in the past 10 years in both sexes. These favorable trends were driven largely by decreases in mortality for three leading cancers in the elderly men [lung, stomach and colorectal cancer (CRC)] and for two of the three most common cancers in the elderly women (stomach and CRC), combined with a leveling off of death rate from lung cancer in women. CONCLUSION: The population-based data in the current study underscore the importance of cancer research and prevention for the older segment in Japan to reduce the additional cancer burden among the growing number of elderly persons.

Three-dimensional optical coherence tomography of Barrett's esophagus and buried glands beneath neosquamous epithelium following radiofrequency ablation.

We report three-dimensional (3D) endoscopic microscopy findings in Barrett's esophagus, using an endoscopic optical coherence tomography (OCT) system in one patient before and in one patient after radiofrequency ablation (RFA). Findings were compared with those in a normal patient without Barrett's esophagus. In the normal patient,findings were of regular flat squamous mucosa with small subepithelial vessels and glands. In the Barrett's esophagus patient, findings were of large, densely packed glands with distortion of mucosal architecture. In the post-RFA case, findings were of a small number of isolated glands buried beneath 300-500 microm of neosquamous epithelium and lamina propria. Neosquamous epithelium is a marker of successful ablative therapy, while buried glands may have potential for dysplastic progression and are difficult to detect using conventional methods. These results indicate a potential role of 3D-OCT endoscopic microscopy for follow-up, including subsurface assessment, of ablative treatments for Barrett's esophagus.